The present invention relates to improved compositions and methods for enzymatic determination of components in biological fluids.
The invention is initially discussed in the context of a creatine phosphokinase assay.
Creatine phosphokinase (CPK) is found primarily in muscle, brain, and heart tissue. Determination of CPK, particularly in blood serum, is one of the most sensitive enzyme assays available for the detection of skeletal muscle disease and is also useful in the diagnosis of myocardial infarction and cerebrovascular incidents.
A basic method for assaying for CPK is the method of C. Oliver, J. Biochem, Volume 61, page 116 (1955). The method has been modified by several workers for use as a diagnostic reagent, see for example, S. B. Rosalki, J. Clin. Lab. Med., Vol. 69, p. 696 (1967). The assay is based on the following principals:
CPK catalyzes the transfer of the phosphate group from creatine phosphate to adenosine diphosphate (ADP) in the presence of magnesium ions and preferably also sulfhydryl groups as activators: EQU Creatine phosphate+ADP CPK Creatine+ATP
where ATP is adenosine phosphate. ATP is used to produce glucose --6-- phosphate from glucose. This reaction is catalyzed by hexokinase (HK): EQU ATP+glucose HK glucose-6-phosphate+ADP
Glucose-6-phosphate is then oxidized by a nicotinamide adenine dinucleotide coenzyme, namely nicotinamide adenine dinucleotide phosphate (NADP) or nicotinamide adenine dinucleotide (NAD) in the presence of glucose-6-phosphate dehydrogenase (G-6-PDH): EQU Glucose-6-phosphate+NAD(P) G-6-PDH 6-phosphogluconate+NAD(P)H
After an initial lag phase, the three reactions proceed stoichiometrically and quantitatively. The NAD(P)H, i.e. reduced NAD or reduced NADP, is determined spectrophotometrically at 340 nm. Alternatively, color coupling reagents may be added to enable spectrophotometric reading in the visible range, e.g. 500 nm.
In the above reaction, the enzymes HK and G-6-PDH may be referred to as indicator enzymes since they are used to convert reaction products into products which are spectrophotometrically measurable. The indicator enzymes in their purified form are used in a portion of about 30 micrograms per 40 milligrams of dry reagent, which is used to make one ml. of liquid reagent. Thus, two problems are presented. The first is that this extremely low percentage, i.e. 0.075% by weight of the dry mixture, is extremely difficult to mix. Therefore, a medium commonly referred to in the art as a bulking agent is needed in which the indicator enzymes may be mixed. Then the indicator enzymes may be uniformly dispersed within a dry mixture so that a conveniently measurable amount of the dry mixture may be used to provide a small amount of the indicator enzymes. This mixing in a medium of greater volume is commonly referred to as bulking. The second problem is that the bulking agent must be suitable for use in both a dry phase of the reagent for storage stability and in the aqueous reagent phase after it is mixed in an aqueous solution for use in assaying.
It is known in the art that it is desirable to provide a kit of several reagents to perform an assay and that providing reagents containing such components as enzymes, coenzymes and/or substrates a material in dry, solid form will be more stable and have a longer shelf-life than a liquid reagent. For example, such advantages are discussed in U.S. Pat. No. 3,540,984 to Alfred Deutsch, issued Nov. 17, 1970. A desired form of preparation of a dry powder enzyme reagent having a long shelf life comprises mixing reagent components in an aqueous solution and lyophilizing them to provide a stable, dry, enzyme-containing material. The enzyme-containing material is in turn mixed with other dry reagent components and with a further dry bulking agent in order to form an economical, conveniently manufactured form of a multicomponent reagent containing compounds which would not be stable over long periods of time in an aqueous phase.
A bulking agent must have the properties normally desirable, i.e. it must be millable, friable and mix well with reagent components. It is also highly desirable that the bulking agent have a salutory effect with respect to enzyme stability. Also, enzyme reactions are subject to interference from many different sources; enzyme activity may be inhibited by anyone of a number of substances. One material that has been used for bulking hexokinase and G-6-PDH is ammonium sulfate. In aqueous solution, this bulking agent ionizes into ammonium ions and sulfate ions. These ions have elevated ionic strength. It has been observed that the activity of CPK is inhibited by various ions such as ammonium and sulfate and by elevated ionic strength in solutions. The problem is therefore presented of stabilizing the CPK reagent in which indicator enzymes are stabilized in dry and in aqueous form without the use of bulking agents which form ions of elevated ionic strength, which may inhibit enzyme activity. In accordance with the present invention, improved bulking is provided for hexokinase and G-6-PDH as well as improved CPK and glucose determinations.
The determination of serum glucose is probably the most frequently performed test in the clinical laboratory, and often utilizes hexokinase and G-6-PDH. Many factors, both physiological and pathological, affect the circulating glucose level. Pathological states which tend to produce hyperglycemia include diabetes mellitus, uremia, hyperthyroidism, and hyperadrenalism. Hypoglycemia is found most commonly with excessive use of insulin and other antidiabetic drugs as well as in certain diseases of the pituitary and adrenals.
One significant commercial glucose determination is a modification of the method of Barthelmai and Czok, Klin. Wschr., Volume 40, page 585 (1962). Glucose is determined by the highly specific hexokinase and glucose-6-phosphate dehydrogenase enzyme system coupled in the final step to the reduction of nicotinamide adenine dinucleotide (NAD), the formation of reduced NAD (NADH) being monitored at 340 nm.
In this method, hexokinase (HK) with a magnesium activator catalyzes the phosphorylation of glucose in the sample by adenosine triphosphate (ATP): ##EQU1## where ADP is adenosine diphosphate.
Glucose-6-phosphate is then oxidized by a nicotinamide adenine dinucleotide coenzyme in the presence of glucose-6-phosphate dehydrogenase (G-6-PDH): EQU glucose-6-phosphate+NAD(P) G-6-PDH 6-phosphogluconate+NAD(P)H
Both reactions proceed stoichiometrically and quantitatively. The NADH produced is determined spectrophotometrically at 340 nm.
Again, it is necessary to bulk and stabilize hexokinase and G-6-PDH. It is also desirable to provide a reagent system or kit in a dry, solid form.
In the quantities of reactive compounds for the above method for 1 ml. of reagent weight about 15 mg. For example, the following components may be used:
0.03 mg. hexokinase PA1 0.03 mg. glucose-6-PDH PA1 0.4 mg. adenosine-triphosphate, sodium salt PA1 0.6 mg. NAD PA1 1.8 mg. magnesium maleate PA1 12 mg. buffer material
These weights of materials cannot be dispensed by commercial equipment with sufficient accuracy. A preferred weight of such a mixture should exceed 70 mg. or preferably 100 mg. Therefore, a dry powder bulking agent to bulk dry reagent components as well as enzyme-containing components is desirable. One such prior bulking agent is mannitol. It is also desirable to provide such a bulking agent which has further useful properties compared to mannitol, such as providing buffering and further contributing to stability. A new such bulking agent, triethanolammonium terephthatale (TEA-TPA), is provided in accordance with the invention.
Another important function in a reagent system is activation or stabilization of enzymes other than the above-defined indicator enzymes. In the present context, activation refers to reversing of oxidation or other adverse effect, while stabilization refers to the prevention thereof. It is important that the enzyme CPK maintain its enzyme activity since its action on the substrate creatine phosphate is necessary for the measurement of CPK in the biological fluid being tested. However, it is known that CPK loses some activity in some sera as a result of reversible inactivation due to the oxidation of essential sulfhydryl groups. This inactivation of CPK may be reversed in part or totally by adding to a reagent composition, and hence reacting with CPK, sulfhydryl-containing compounds such as glutathione, mercaptoacetic acid, or dithiothereitol (DTT). This may be accomplished by adding the compound to the serum or by incorporating it in the enzyme assay mixture. The most commonly used sulfhydryl compounds for this application are glutathione (GSH) and DTT.
However, it has been noticed that in the embodiment in which iodonitrotetrazolium violet (INT) coupling is provided for spectrophotometric measurement in the visible range, sulfhydryl compounds slowly reduce the INT to form its colored formazan. This increases the amount of background color, or may be said to increase the blank reaction. The range of the useful curve of optical absorbance versus concentration of CPK which is useful is thereby reduced. It should be noted that GSH and DTT in particular are quite expensive. GSH has also been criticized somewhat in the literature. For example, see G. Anido, S. B. Rosalki, E. J. van Kampen and M. Ruben, Quality Control in Clinical Chemistry, pp. 180-183, (Walter de Gruyter, Berlin, 1975). It is stated that definitive recommendations on the appropriate thiol cannot be made.
It is therefore desirable to find an activator for CPK which is also compatible with the other components in the reagent system. It is also preferable if that reagent is of lower cost. Another consideration is that the activator be useful in a dry, solid reagent system having a long shelf-life. The material selected must be useful in the initial reagent preparation, the dry phase, and again in the aqueous state when the reconstituted reagent is used in the laboratory. It is also desirable to provide an improved stabilizer for enzymes used for the determination of glucose and serum urea nitrogen. Such an activator stabilizer is provided in accordance with the present invention.
The determination of serum urea nitrogen, also often referred to as BUN, is widely used for evaluation of the kidney function. One standard method is that of H. Talke and G. E. Schubert, Klin.-Wschr. Vol. 43,p. 174 (1965). This enzymatic method does not require the use of corrosive reagents or high reaction temperatures.
This determination is based on the following principles:
Urea is hydrolyzed by urease: EQU (NH.sub.2).sbsb.2CO+H.sub.2 O .sup.urease 2NH.sub.3 +CO.sub.2
Ammonia is produced which aminates .alpha.-ketoglutarate in the presence of glutamate dehydrogenase (GLDH) with concurrent oxidation of NADH: EQU NH.sub.3 +.alpha.-ketoglutarate+NADH.fwdarw.glutamate+H.sub.2 O+NAD
Both reactions proceed stoichiometrically and quantitatively. The disappearance of NADH is measured at 340 nm spectrophotometrically.
The novel activator and stabilizer of the present invention interacts in the CPK and glucose determinations described above and also interacts with the improved bulking agents referred to above to provide further improved CPK and glucose determinations. An improved serum urea nitrogen determination and improved stabilization of enzymes therein are also provided.
All of the elements described above cooperate to form an interactive reagent system. This concept is illustrated and defined by the example of the novel glucose reagent system. A novel bulking agent is provided for bulking indicator enzymes. This bulking agent improves stability in the dry phase. A further novel bulking agent is also provided for bulking other dry reagent components and also contributes to stability in the dry phase. The two bulking agents both contribute to stability while neither adversely affects the other. A novel enzymes stabilizer is also included in the dry reagent system. This stabilizer acts in the aqueous phase to improve reagent stability. The stabilizer does not adversely affect stability in the dry phase, and the bulking agents do not adversely affect stability in the aqueous phase. Also, the further bulking agent acts as a buffer in the aqueous phase. The bulking agents do not interfere with stability or with the chemical reactions in the aqueous phase. Compatability of reagent system components contributes to the desirable result of providing a reagent system which may be stored by a user on a shelf for prolonged periods of time before use and which is also stable for long periods of time in use. (The terms "prolonged" and "long" are used in their well-known sense in the context of clinical chemistry laboratory use.)
Stability is a significant aspect in the commercial use of reagents. A reagent with a long shelf life may remain in distribution channels such as in a manufacturer's and distributor's inventory before shipment to a laboratory, and the laboratory may order a sufficiently large inventory so that frequent reordering is not necessary. Increasingly critical cost factors are thus somewhat alleviated. In vitro diagnostic reagents must be discarded after expiration dates based on their stability, and it is important to provide a reagent which a laboratory will not need to return or discard before use.